Alzheimer's Disease is a progressive neurodegenerative disorder that leads to the death of brain cells that cannot be replaced once lost. The two key neuropathological hallmarks of AD are the presence of senile plaques predominantly comprised of aggregated beta-amyloid protein (Aβ) and neurofibrillary tangles (NFTs) formed by the accumulation of hyperphosphorylated tau protein. Currently, the clinical diagnosis of AD requires an evaluation of medical history and physical examination including neurological, neuropsychological and psychiatric assessment, as well as various biological, radiological and electrophysiological tests. Despite the battery of tests, a definitive diagnosis can only be achieved by post-mortem brain examination. Therefore, there is an unmet need for a simple biochemical test that can detect AD at an early stage, monitor progression of the disease, and discriminate between AD, normal, non-AD dementias and other neurological disorders.
Three different biomarkers in Cerebrospinal Fluid (CSF) have been particularly well researched: neuronal thread protein, tau and derivatives of amyloid precursor protein (APP).
Neuronal thread protein is known to be overexpressed in brain neurons in AD patients. The company NYMOX has developed a quantitative test for measuring levels of a specific type of neuronal thread protein (AD7c-NTP) in CSF and urine. Quite a number of studies have evaluated CSF-tau as an ante-mortem marker for AD mainly using enzyme-linked immunoabsorbent assays (ELISA) as the measurement assay. In most of these studies, total tau has been measured although there is an increasing body of literature also describing the analysis of phosphorylated variants of the same protein involved in the formation of NFTs. ELISAs that can distinguish between the major form of Aβ ending at amino acid 40 (Aβ40) and the senile plaque forming species ending at position 42 (Aβ42) have also been developed and evaluated extensively for CSF analysis. All three assays, either used individually, or in the case of tau and Aβ42, in combination, do not have the required sensitivity and specificity values for routine clinical use, particularly for early diagnosis and discrimination between AD and other non-AD dementias. In addition, attempts to measure tau and Aβ42 in blood have been met with limited success, further restricting their widespread adoption into clinical practice.
A wide spectrum of other aberrations, other than NTP, Tau and Aβ, has been reported in AD patient CSF. Many of the identified (protein sequence confirmed) CSF markers reported herein have been shown to be either increased or decreased in AD patients versus normal individuals. For example, the protein Ubiquitin is known to complex with hyperphosphorylated Tau during maturation of NFTs in the brains of AD patients (Iqbal et. al. J Neural Transm Suppl. 53:169-80 (1998)). Ubiquitin levels in CSF of AD and neurological control groups have been shown to be significantly higher than those of non-neurological aged controls (Wang et. al. Acta Neuropathol (Berl). 82(1):6-12 (1991); Kudo et. al. Brain Res. 639(1):1-7 (1994)).
The acute phase/inflammatory protein alpha(1)-antichymotrypsin (ACT) is overproduced in the AD brain. ACT also can promote the formation of, and is associated with, neurotoxic amyloid deposits (Potter et. al. Neurobiol Aging. 22(6):923-30 (2001)). The levels of ACT in both serum and CSF are significantly and specifically higher in patients with Alzheimer-type dementia than in control subjects (Matsubara et. al. Ann Neurol. 28(4):561-7(1990)). There is a particularly close association of increases in CSF-ACT with late onset AD (Harigaya et. al. Intern Med. 34(6):481-4 (1995)).
Chromogranin A (CrA) is the major protein of large dense-core synaptic vesicles and may be of value as a biochemical marker for synaptic function in AD. One report described no difference between the AD, vascular dementia, and age-matched control groups except when comparing a familial subtype (AD Type I) with controls where there was a statistically significant elevation of CSF CrA in the diseased individuals (Blennow et. al. Dementia. 6(6):306-11 (1995)).
Beta-2-Microglobulin (B2M) is an initiator of inflammatory responses modulated by interferons and certain cytokines (Hoekman et. al. Neth. J. Med. 28:551-557 (1985)). A proteome analysis of CSF by two-dimensional electrophoresis (2D-gel) has shown a significant increase of B2M in AD patients (Davidsson et al., Neuroreport, 13:611-615 (2002)), and more recently these results were confirmed by SELDI analysis (Carrette, O. et. al., Proteomics, 3:1486-1494 (2003)).
Transthyretin (TTR) has been shown to interact with Aβ, possibly preventing amyloid formation in biological fluids and in the brain. (Tsuzuki et al., Neurosci Lett, 10:171-174 (2000)). One identified TTR isoform was shown to be increased in AD-CSF using 2D gel analysis of a small number of AD and control patients (Davidsson, supra.). However, this result conflicts with other reports showing a clear decrease of TTR in CSF from AD patients compared with controls (Serot et. al. J Neurol Neurosurg Psychiatry. 63(4):506-8 (1997); Riisoen et. al. Acta Neurol Scand. 78(6):455-9 (1998)). This decrease is also negatively correlated with the senile plaque (SP) abundance (Merched et. al. FEBS Lett. 425(2):225-8 (1998)).
Cystatin C, a cysteine protease inhibitor, has been implicated in the neurodegenerative and repair processes of the nervous system, and the deposition of the same protein together with beta amyloid peptide was found as cerebral amyloid angiopathy (CAA) in different types of dementias (Levy et. al. J. Neuropathol. Exp. Neurol. 60:94-104). Full length Cystatin C was found as a CSF marker for AD in a previous SELDI profiling study (Carrette, supra.). A relative blood-brain barrier (BBB) dysfunction is associated with AD among very elderly individuals. The CSF/serum albumin ratio can be used as a measure of BBB function. Mean CSF/serum albumin ratio has been reported to be higher in all dementias studied, including AD, than in nondemented individuals (Skoog et al, Neurology. 50:966-71 (1998)).
Transferrin (TF) plays a role in anti-oxidant defense in serum and is also produced in the brain where its role in oxidative stress is unclear. A study on Down's syndrome patients suffering from progressive dementia showed decreased levels of TF when compared to age-matched controls with no neurological disease (Elovaara Acta Neurol Scand. 69(5):302-5(1994)).
Prior studies evaluating the different biochemical markers in the CSF of dementia patients have employed assay methods, such as ELISA, that use indirect means of measuring the analyte of interest. These methods are not capable of discerning the different processed forms of proteins revealed other processes. Furthermore, traditional assay methods have left unexplored avenues of treatment relating to the different processed forms of proteins. Thus, a need exists for methods which diagnosis Alzheimer's disease before significant neuronal loss has occurred, and for therapeutic treatments to prevent progression of the disease.